Novel recombinant bi-functional fusion protein and preparation and application therefor

ABSTRACT

The present invention provides a recombinant fusion protein containing a first extracellular Ig-like domain of a signal-regulator protein alpha (SIRPα), linked to an Fc fragment of a human IgG1. The present invention also provides a polynucleotide encoding the recombinant fusion protein, an expression vector containing the polynucleotide, a method for producing the recombinant protein and a method for treating a disease caused by over expression of CD47.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/566,724 filed Oct. 16, 2017, now U.S. Pat. No. 10,800,821, which is aU.S. National Stage Application of PCT/CN2015/094739 filed Nov. 16,2015, which claims priority to Chinese patent application Serial No.CN201510203619.7 filed Apr. 24, 2015, the entire specification of eachbeing incorporated herein by reference.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said ASCII copy, is named 00025SL.txt and is 22.7 kbytes insize.

FIELD OF THE INVENTION

The invention relates to a recombinant bi-functional fusion protein,preparation and use thereof, especially its use in tumor therapies.

BACKGROUND OF THE INVENTION

Cancer cells have developed some mechanisms to evade a host's immunesurveillance so that they can grow faster. Among the mechanisms, thefollowing three are well known. 1) To evade immune surveillance byT-lymphocytes. The cancer cells usually express a high level of membraneprotein PD-L1 and PD-L2, both of which bind to PD-1 on the surface ofT-cell, inducing T-cell apoptosis. 2) To evade immune surveillance bynatural killer (NK) cells. The NKG2D protein on the surface of NK cells,upon binding to the MICA/MICB proteins on the surface of the cancercells, can activate NK cells to kill the cancer cells. However, cancercells have developed a mechanism that promotes the detachment ofMICA/MICB from the cell membranes. The detached MICA/MICB binds to theNKG2D on the surface of NK cells, blocking the binding of NKG2D toMICA/MICB on the surface of cancer cells. 3) To evade the immunesurveillance by macrophages (Mφ). Almost all cancer cells express ontheir surfaces a high level of CD47, which binds to the signalregulatory protein (SIRPα) on the surface of Mφ, thereby inducing theproduction of an inhibitory signal, which inhibits the phagocytosis ofcancer cells by Mφ. It can be seen that the cancer cells are quite“smart” and reproduce quickly depending on their developed evasionmechanisms. Accordingly, development of effective anti-cancer drugs forkilling all the cancer cells needs to target these mechanisms.

The present invention is directed to the signal regulatory proteinSIRPα. The signal regulatory protein (SIRP) is a transmembraneglycoprotein, including three family members, SIRPα (CD172a), SIRPβ(CD172b) and SIRPγ (CD172g). The three proteins have similarextracellular regions but distinct cytoplasmic regions (FIG. 1A). Theextracellular region contains three immunoglobulin (Ig) domains—oneIgV-set and two IgC-set domains. The cytoplasmic region of SIRPα(CD172a) contains two domains which transmit inhibitory signals toinhibit the corresponding function(s) of the cell. SIRPβ (CD172b) andSIRPγ (CD172g) have very short cytoplasmic regions without anysignal-transmitting domain. However, SIRPβ (CD172b) may transmit theactivating signals through its association with the adaptor proteinssuch as DAP12 (FIG. 1). SIRPs are mainly expressed in macrophages (Mφ),dendritic cells (DCs) and neurons.

CD47 is also a transmembrane glycoprotein belonging to theimmunoglobulin superfamily, and is expressed on the surface of all celltypes including red blood cells. Ligands for CD47 include integrins,thrombospondin-1 and SIRPs. CD47 has many biological functions,including in cell migration, activation of T-cells and DCs, and axondevelopment. In addition, CD47, by interacting with SIRPα, can inhibitthe phagocytosis by macrophages. By emitting a “don't eat me” signal,CD47 protects normal cells, such as blood cells, from being attacked bymacrophages.

Studies have shown that many tumor or cancer cells over-express CD47,which, by binding to the SIRPα on the cell surface of macrophages,prevent phagocytosis of the cancer cells by macrophages. This is deemedas one of the mechanisms adopted by tumors to evade the host's immunesurveillance. Cancers that over-express CD47 include acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphoblasticleukemia (ALL), non-hodgkins lymphoma (NHL), multiple myeloma (MM),bladder cancer, ovarian cancer, prostate cancer, lung cancer, coloncancer, breast cancer, and pancreatic cancer.

Other studies showed that injection of CD-47 specific antibody thatblocks the binding of CD47 to SIRPα can significantly inhibit tumorgrowth in tumor-bearing mice. Tumor or cancer cells were eliminatedcompletely when the same antibody was injected into the mice carryinghuman leukemia cells (Theocharides A P A, et al., 2012).

An Fc receptor is a protein found on the surface of certain cells,including B lymphocytes, follicular dendritic cells, natural killercells, macrophages, neutrophils, eosinophils, basophils, and mast cells.These cells contribute to the protective functions of the immune system.The Fc receptor binds to antibodies that are attached to infected cells,invading pathogens or cancer cells, and stimulates phagocytic orcytotoxic cells to destroy microbes, infected cells or cancer cells byantibody-mediated phagocytosis or antibody-dependent cell-mediatedcytotoxicity.

The binding activity of SIRPα-Fc or SIRPαD1-Fc to CD47 has been reported(Lee W Y et al., 2007), but both having insufficient affinity to CD47.

SUMMARY OF THE INVENTION

The present invention discloses a recombinant fusion protein capable ofeliminating tumors by i) blocking tumor cell induced production ofinhibitory signals to macrophages, and ii) directly stimulatingphagocytosis by macrophages. The fusion protein comprises a firstextracellular Ig-like domain of human Signal-regulatory protein alpha(SIRPα) (SIRPαD1) linked to an Fc fragment of human IgG1 (SIRPαD1-Fc).The fusion protein is a homodimer having a molecular weight of 90 kDa,consisting of a first extracellular domain (Domain 1, D1) of SIRPαlinked to an Fc fragment of human IgG (SIRPαD1-Fc). The fusion proteinhas a better binding activity to targets, compared to a recombinantprotein containing the entire extracellular region of SIRPα (SIRPα-Fc).The present inventors have further discovered that the binding activityto targets can be further improved when the site for glycosylation inSIRPαD1 is removed (N89A); and the binding activity of Fc to Fcreceptors may significantly improve SIRPαD1-Fc's anti-tumor activity.

A stably-expressing cell line of Chinese hamster ovary (CHO) cells hasbeen obtained by screening, and proteins are produced by culturing thecell line in a shaking bed. In vitro experiments have shown that theprotein of the present invention may bind to CD47 with an evidentlyimproved binding activity compared to SIRPα-Fc. If the site forglycosylation “NIT” in SIRPαD1 is removed by genetic engineering (N89A)(SIRPαD1-Fc (N89A), designated as HY03M), the binding activity to CD47is improved. In another aspect, when the 192^(th) amino acid residue inthe Fc region is converted from aspartic acid to alanine (D192A)(SIRPαD1-Fc (N89A/D192A), designated as HY03MM), the binding activity toFc receptors (CD16a, CD32, CD64) is evidently decreased. The in vivoanti-tumor effect of HY03M has been studied using human acutelymphoblastic leukemia and acute promyelocytic leukemia-carrying mousemodels, indicating that HY03M has extremely good anti-tumor activity.Tumor growth is completely inhibited in mice treated by HY03M, and tumoris no longer detected in some mice. To confirm Fc's contribution to theanti-tumor activity, the in vivo therapeutic effects of HY03M and HY03MMhave been tested in mice carrying human lymphoma in comparison toRituximab. It turns out that HY03M has an evident anti-tumor effectcompared to the negative control group, and its effect is also muchbetter than Rituximab. Despite of a good inhibitory effect on tumorgrowth, HY03MM's activity is inferior to HY03M, suggesting that the Fcregion is involved in tumor inhibition by binding to Fc receptors onmacrophages.

In one embodiment, the recombinant protein of the present invention isHY03M (SEQ ID NO.: 5, SEQ ID NO.: 6). HY03M has a high anti-tumoractivity, and inhibits tumor growth by i) blocking interaction betweenCD47 and SIRPα; and/or ii) activating macrophages by binding Fc to Fcreceptors. HY03M may treat various CD47+ tumors.

The present invention also provides a nucleic acid molecule encoding therecombinant bi-functional fusion protein and an expression vectorexpressing the protein, a method for producing the protein and a methodfor treating a disease over-expressing CD47.

In one embodiment, the recombinant bi-functional fusion protein of thepresent invention comprises an extracellular Ig-like domain ofSignal-regulatory protein (SIRP), linked to an Fc fragment of humanIgG1, wherein the protein binds to CD47 to block the binding of CD47 toSIRP on the surface of macrophages so as to stimulate phagocytosis oftumor cells by macrophages.

In one embodiment, the signal-regulatory protein in the recombinantbi-functional fusion protein is SIRPα, and the extracellular Ig-likedomain of the signal-regulatory protein is SIRPαD1.

In one embodiment, the recombinant bi-functional fusion protein of thepresent invention comprises an Fc fragment, a part of an immunoglobulinmolecule. Although an Fc fragment does not have any antigen-bindingsite, but does stimulate effector functions. For example, the Fcfragment facilitates the binding of the antibody with Fc receptors orcomplement proteins. In one embodiment, the Fc fragment is an Fcfragment of IgG1.

In one embodiment, the recombinant bi-functional fusion protein of thepresent invention comprises an amino acid sequence having at least 95%identity to SEQ ID NO.: 6. In one embodiment, the recombinantbi-functional fusion protein of the present invention comprises an aminoacid sequence having at least 98% identity to SEQ ID NO.: 6. In oneembodiment, the recombinant bi-functional fusion protein of the presentinvention comprises an amino acid sequence having at least 99% identityto SEQ ID NO.: 6. In one embodiment, the recombinant bi-functionalfusion protein of the present invention comprises an amino acid sequenceset forth in SEQ ID NO.: 6.

In one embodiment, the present invention provides a polynucleotidemolecule encoding the recombinant bi-functional fusion protein of thepresent invention, wherein the recombinant bi-functional fusion proteincomprises an extracellular Ig-like domain of signal-regulatory protein(SIRP), linked to an Fc fragment of human IgG1, wherein the proteinbinds to CD47 to block the binding of CD47 to SIRP on the surface ofmacrophages so as to stimulate phagocytosis of tumor cells bymacrophages.

In one embodiment, the polynucleotide molecule of the present inventionencodes a recombinant bi-functional fusion protein comprising SIRPα,preferably SIRPαD1.

In one embodiment, the polynucleotide molecule of the present inventionencodes a recombinant bi-functional fusion protein comprising an Fcfragment of an Immunoglobulin, preferably an Fc fragment of human IgG1.

In one embodiment, the polynucleotide molecule of the present inventionencodes a recombinant bi-functional fusion protein having an amino acidsequence that is at least 95% identical to SEQ ID NO.: 6. In oneembodiment, the polynucleotide molecule of the present invention encodesa recombinant bi-functional fusion protein having an amino acid sequencethat is at least 98% identical to SEQ ID NO.: 6. In one embodiment, thepolynucleotide molecule of the present invention encodes a recombinantbi-functional fusion protein having an amino acid sequence that is atleast 99% identical to SEQ ID NO.: 6. In one embodiment, thepolynucleotide molecule of the present invention encodes a recombinantbi-functional fusion protein having an amino acid sequence set forth inSEQ ID NO.: 6.

In one embodiment, the present invention provides an expression vectorcomprising the polynucleotide molecule of the present invention, thepolynucleotide molecule encoding a recombinant bi-functional fusionprotein.

In one embodiment, the present invention provides a host cell comprisingthe expression vector of the present invention.

In one embodiment, the present invention provides a pharmaceuticalcomposition, comprising the recombinant bi-functional fusion protein ofthe present invention, and at least one adjuvant.

In one embodiment, the present invention provides a method for treatinga disease caused by over-expression of CD47, comprising administering toa patient or a subject a therapeutically effective amount of thepharmaceutical composition of the present invention. In one embodiment,the present invention provides the use of the recombinant bi-functionalfusion protein in the manufacture of a pharmaceutical composition fortreatment of a disease caused by over-expression of CD47.

In one embodiment, the method of the present invention is for treating adisease selected from the group consisting of acute myelocytic leukemia(AML), chronic myelocytic leukemia (CML), acute lymphoblastic leukemia(ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), Bladdercancer, ovarian cancer, prostate cancer, lung cancer, colon cancer,breast cancer, pancreatic cancer, and renal cell carcinoma. In oneembodiment, the present invention provides a method for treating Crohn'sdisease, allergic asthma and rheumatoid arthritis.

DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D is a schematic diagram of the structures and mechanism ofaction of SIRPs and SIRPαD1.

FIG. 2A-2B shows nucleic acid sequence and amino acid sequence ofSIRPα-Fc.

FIG. 3A-3B shows nucleic acid sequence and amino acid sequence ofSIRPαD1-Fc.

FIG. 4A-4B shows nucleic acid sequence and amino acid sequence of HY03M.

FIG. 5A-5B shows nucleic acid sequence and amino acid sequence ofHY03MM.

FIG. 6A-6D shows SDS-PAGE analysis of four fusion proteins.

FIG. 7A-7B shows the binding activities of SIRPα-Fc and SIRPαD1-Fc tothe targets on PC-3 cells, respectively.

FIG. 8 shows the blockade of fluorescently labeled targets by unlabeledproteins.

FIG. 9A-9B shows the binding activity to Fc receptors and the inducedphagocytosis of tumor cells by macrophages.

FIG. 10A-10B shows the in vivo therapeutic efficacies of four fusionproteins on tumors, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a recombinant fusion protein, whichcan eliminate tumors via two approaches, i.e., to block tumor cellinduced production of inhibitory signals to macrophages, and to directlystimulate the phagocytosis by macrophages. The protein consists of afirst extracellular Ig-like domain of human Signal-regulatory proteinalpha (SIRPα) (SIRPαD1) linked to an Fc fragment of humanIgG1(SIRPαD1-Fc). The fusion protein is a homodimer having a molecularweight of 90 kDa. A stably-expressing cell line of Chinese hamster ovary(CHO) cells has been obtained by screening, and 100 mg of the proteinsare produced by culturing the cell line in a shaking bed. In vitroexperiments have shown that the proteins may bind to CD47 with anevidently improved binding activity compared to SIRPα-Fc. If the sitefor glycosylation (“NIT”) in SIRPαD1 is removed by genetic engineering(N89A) (SIRPαD1-Fc (N89A), designated as HY03M), the binding activity toCD47 will be improved. In another aspect, when the 211^(th) amino acidresidue in the Fc region is converted from aspartic acid to alanine(D192A) (SIRPαD1-Fc (N89A/D192A), designated as HY03MM), the bindingactivity to Fc receptors (CD16a, CD32, CD64) is evidently decreased. Thein vivo anti-tumor effect of HY03M has been studied using human acutelymphoblastic leukemia and acute promyelocytic leukemia-carrying mousemodels, indicating that HY03M has extremely good anti-tumor activity.Tumor growth is completely inhibited in mice treated by HY03M, and tumoris no longer detected in some mice. To confirm Fc's contribution to theanti-tumor activity, in vivo therapeutic efficacies of HY03M and HY03MMhave been tested in mice carrying human lymphoma in comparison toRituximab. It turns out that HY03M has an evident anti-tumor effectcompared to the negative control group, and its effect is also muchbetter than Rituximab. Despite of a good inhibitory effect on tumorgrowth, HY03MM's activity is inferior to HY03M, suggesting that the Fcregion is involved in tumor inhibition by binding to Fc receptorsdistributed on macrophages.

The fusion protein of the present invention contains two fragments,i.e., the target-binding fragment (SIRPαD1) and the Fc fragment.

The recombinant bi-functional fusion protein of the present inventionmay further bind to non-polypeptide molecules for the purpose ofconferring desired properties such as reducing degradation and/orincreasing half-life, reducing toxicity, reducing immunogenicity, and/orincreasing the biological activity. Exemplary molecules include but arenot limited to polymers such as polyethylene glycol (PEG), polylysine, adextran; a lipid; a cholesterol group (such as a steroid); acarbohydrate, or an oligosaccharide molecule.

The Fc fragment may be 232 amino acids in size, and comprises a cysteinin the hinge region, two cysteins in the CH2 region and two cysteins inthe CH3 region. The cystein in the hinge region contributes to theformation of disulfide bond between two monomers, thereby generating ahomodimer, while the cysteins in the CH2 and CH3 regions can formintrachain disulfide bonds to stabilize the protein.

For example, the extracellular Ig domain of SIRP (SIRPα, SIRPγ) capableof binding CD47 can be used in the fusion protein.

Preferably, human-derived sequences may be used in human cancertherapies, as the strong immunogenicity of the proteins or peptides fromnon-human animals may lead to allergy and other adverse effects.However, other animal proteins or peptides may also be used in thepresent invention based on different application purposes.

In one embodiment, the signal-regulatory protein in the recombinantbi-functional fusion protein of the present invention is SIRPα. Theextracellular Ig-like domain of the signal-regulatory protein isSIRPαD1.

In the present invention, the Fc fragment from any immunoglobulin can beused. Immunoglobulins include IgG, IgA, IgM, IgD and IgA, among whichIgG is the most abundant and relatively stable. The Fc fragment of IgG,the Fc fragment of IgG1 and the Fc fragment of human IgG1 are preferredin the present invention as these Fc fragments exhibit the highestbinding activity with staphylococcus Protein A and therefore can beeasily purified.

In one embodiment, the recombinant bi-functional fusion protein of thepresent invention comprises a first extracellular Ig-like domain ofhuman SIRPα, linked to an Fc fragment of human IgG1.

In one embodiment, the amino acid sequence of the recombinantbi-functional fusion protein of the present invention is shown in FIG.4B (SEQ ID NO.: 6). In another embodiment, the polypeptide has an aminoacid sequence set forth in SEQ ID NO.: 6. In another embodiment, thepolypeptide has an amino acid sequence with at least 80%, 85%, 90%, 95%,98% or 99% identity to SEQ ID NO.: 6, wherein the polypeptide is capableof binding to CD47 and is able to inhibit tumor cell growth.

In another aspect, the present invention provides an isolated nucleicacid molecule comprising a polynucleotide encoding a recombinantbi-functional fusion protein, wherein, the recombinant bi-functionalfusion protein comprises a polypeptide having the sequence set forth inSEQ ID NO.: 6. In one embodiment, the polypeptide has an amino acidsequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQID NO.: 6, wherein the polypeptide is capable of binding to CD47 and isable to inhibit tumor cell growth.

The present invention also discloses a pharmaceutical compositioncomprising the aforementioned recombinant bi-functional fusion protein,and at least one pharmaceutically acceptable excipient. If needed, oneor more pharmaceutically acceptable carriers or excipients may also beincluded in the pharmaceutical composition. The carriers includediluents, excipients, bulking agents, bonding agents, wetting agents,disintegrating agents, absorption enhancers, surfactants, sorptioncarriers, lubricants and the like ordinary in the pharmaceutics.

Such compositions comprise a therapeutically or prophylacticallyeffective amount of the polypeptide or protein in admixture withpharmaceutically acceptable materials, and physiologically acceptableformulation materials. The pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, odor, sterility,isotonicity, stability, rate of dissolution or release, adsorption orpenetration of the composition. Suitable formulation materials include,but are not limited to, amino acids (such as glycine, glutamine,asparagine, arginine or lysine); antimicrobials; antioxidants (such asascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (suchas borate, ammonium bicarbonate, Tris-HCl, citrates, phosphates, andother organic acids); bulking agents (such as mannitol or glycine),chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides; disaccharides and other carbohydrates (such as glucose,mannose, or dextrins); proteins (such as serum albumin, gelatin orimmunoglobulins); coloring agents; flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as PLURONICS® or poloxamers, PEG,sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80,TRITON® X (Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenylether), tromethamine, lecithin, cholesterol, tyloxapal); stabilityenhancing agents (sucrose or sorbitol); tonicity enhancing agents (suchas alkali metal halides, preferably sodium or potassium chloride,mannitol sorbitol); delivery vehicles; diluents; excipients and/orpharmaceutical adjuvants.

The optimal pharmaceutical composition will be determined by one skilledin the art depending upon, for example, the intended route ofadministration, delivery format, and desired dosage. See for example,Remington's Pharmaceutical Sciences, supra. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the polypeptide. For example, suitablecompositions may be water for injection and physiological salinesolution.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in injection. For example, the vehicle or carrier maybe neutral buffered saline or saline mixed with serum albumin. Otherexemplary pharmaceutical compositions comprise Tris buffers, or acetatebuffers, which may further include sorbitol or a suitable substitutethereof. In one embodiment of the present invention, compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the therapeutic composition may be formulatedas a lyophilizate using appropriate excipients such as sucrose.

The formulations can be delivered in a variety of methods, for example,by inhalation therapy, orally, or by injection. When parenteraladministration is contemplated, the therapeutic compositions for use inthis invention may be in the form of a parenterally acceptable aqueoussolution comprising the desired polypeptide in a pharmaceuticallyacceptable vehicle. A particularly suitable vehicle for parenteralinjection is sterile distilled water in which a polypeptide isformulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (polylactic acid, polyglycolic acid), beads, orliposomes, that provides for the controlled or sustained release of theproduct which may then be delivered via a depot injection. Hyaluronicacid may also be used, and this may have the effect of promotingsustained duration in the circulation. Other suitable means for theintroduction of the desired molecule include implantable drug deliverydevices.

In another aspect, pharmaceutical formulations suitable for injectableadministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions. In anotherembodiment, a pharmaceutical composition may be formulated forinhalation. Inhalation solutions may also be formulated with apropellant for aerosol delivery, in yet another embodiment, solutionsmay be nebulized. Pulmonary administration is further described in PCTApplication No. PCT/US94/001875, which describes pulmonary delivery ofchemically modified proteins.

It is also contemplated that certain formulations may be administeredorally. In one embodiment of the present invention, molecules that areadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule may be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the therapeutic molecule. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders may also beemployed. Pharmaceutical compositions for oral administration can alsobe formulated using pharmaceutically acceptable carriers well known inthe art in dosages suitable for oral administration. Such carriersenable the pharmaceutical compositions to be formulated as tablets,pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions,and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arable and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations that can be used orally also includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving polypeptides in sustained-or controlled-delivery formulations. Techniques for formulating avariety of other sustained- or controlled-delivery means, such asliposome carriers, bio-erodible micro-particles or porous beads anddepot injections, are also known to those skilled in the art. See forexample, PCT/US93/00829 that describes controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. Additional examples of sustained-release preparationsinclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices may includepolyesters, hydrogels, polylactides (Langer et al, J. Biomed. Mater.Res., 15: 167-277, (1981); Langer et al., Chem. Tech., 12:98-105(1982)), ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., PNAS (USA),82:3688 (1985); EP 36,676; EP 88,046; EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically must be sterile. This may be accomplished by filtrationthrough sterile filtration membranes. Where the composition islyophilized, sterilization using this method may be conducted eitherprior to or following lyophilization and reconstitution. The compositionfor parenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or a dehydrated or lyophilized powder. Such formulations may be storedeither in a ready-to-use form or in a form (e.g., lyophilized) requiringreconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits may each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

An effective amount of a pharmaceutical composition to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment will thus vary depending, inpart, upon the molecule delivered, the indication for which thepolypeptide is being used, the route of administration, and the size(body weight, body surface or organ size) and condition (the age andgeneral health) of the patient. Accordingly, the clinician may titer thedosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical dosage may range from about 0.1 mg/kg toup to about 100 mg/kg or more, depending on the factors mentioned above.Polypeptide compositions may be preferably injected or administeredintravenously. Long-acting pharmaceutical compositions may beadministered every three to four days, every week, or biweekly dependingon the half-life and clearance rate of the particular formulation. Thefrequency of dosing will depend upon the pharmacokinetic parameters ofthe polypeptide in the formulation used. Typically, a composition isadministered until a dosage is reached that achieves the desired effect.Tine composition may therefore be administered as a single dose, or asmultiple doses (at the same or different concentrations/dosages) overtime, or as a continuous infusion. Further refinement of the appropriatedosage is routinely made. Appropriate dosages may be ascertained throughuse of appropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebro ventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional routes, intramedullary, intrathecal,intraventricular, transdermal, subcutaneous, or intraperitoneal; as wellas intranasal, enteral, topical, sublingual, urethral, vaginal, orrectal means, by sustained release systems or by implantation devices.Where desired, the compositions may be administered by bolus injectionor continuously by infusion, or by implantation device. Alternatively oradditionally, the composition may be administered locally viaimplantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Wherean implantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of the desired molecule may bevia diffusion, timed-release bolus, or continuous administration.

In some cases, the bi-functional fusion protein of the present inventioncan be delivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptide. Such cells may be animal or human cells, andmay be autologous, heterologous, or xenogeneic. Optionally, the cellsmay be immortalized. In order to decrease the chance of an immunologicalresponse, the cells may be encapsulated to avoid infiltration ofsurrounding tissues. The encapsulation materials are typicallybiocompatible, semi-permeable polymeric enclosures or membranes thatallow the release of the polypeptide product(s) but prevent thedestruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

A gene therapy in vivo is also envisioned wherein a nucleic acidmolecule encoding the bi-functional fusion protein of the presentinvention, or a derivative thereof is introduced directly into thesubject. For example, a nucleic acid sequence encoding a bi-functionalfusion protein of the present invention is introduced into target cellsvia local injection of a nucleic acid construct with or without anappropriate delivery vector, such as an adeno-associated virus vector.Alternative viral vectors include, but are not limited to, retroviruses,adenovirus, herpes simplex virus and papilloma virus vectors. Physicaltransfer of the virus vector may be achieved in vivo by local injectionof the desired nucleic acid construct or other appropriate deliveryvector containing the desired nucleic acid sequence, liposome-mediatedtransfer, direct injection (naked DNA), or microparticle bombardment(gene-gun).

The compositions of the present disclosure may be used alone or incombination with other therapeutic agents to enhance their therapeuticeffects or decrease potential side effects.

Another object of the present invention is to provide a method forpreparing the above recombinant bi-functional fusion protein and thepharmaceutical composition comprising the same. In one embodiment, themethod comprises (1) providing an protein-encoding polynucleotidemolecule; (2) constructing an expression vector comprising thepolynucleotide molecule of (1); (3) transfecting or transformingsuitable host cells with the expression vector of (2) and cultivatingthe host cells to express the protein; and (4) purifying the protein.The preparation may be carried out with well-known technologies by anordinarily skilled artisan.

Another object of the present invention is to provide a method oftreating cancer using the pharmaceutical composition of the presentinvention, comprising administrating an effective amount of theaforementioned pharmaceutical composition to the patients or subjects inneed thereof. In one embodiment, the pharmaceutical composition is usedto treat CD47-overexpressing tumors or cancers, including but notlimited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML),acute lymphoblastic leukemia (ALL), non-hodgkins lymphoma (NHL),multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer,lung cancer, colon cancer, breast cancer, pancreatic cancer and renalcancer.

In one embodiment, the diseases related to over-expression of CD47include but are not limited to Crohn's disease, allergic asthma, andrheumatoid arthritis.

Also, the present invention provides a polynucleotide molecule encodingthe recombinant bi-functional fusion protein and an expression vectorexpressing the recombinant bi-functional fusion protein. Examples ofvectors include but are not limited to plasmids, viral vectors, yeastartificial chromosomes (YACs), bacterial artificial chromosomes (BACs),transformation-competent artificial chromosomes (TACs), mammalianartificial chromosomes (MACs) and human artificial episomal chromosomes(HAECs).

The present invention provides host cells comprising the aboveexpression vectors. The host cells may be transformed or transfectedwith the expression vectors. Suitable host cells include Escherichiacoli, yeasts and other eukaryotes. Preferably, Escherichia coli, yeastor mammalian cell lines (such as COS or CHO) are used.

The present invention is now further described with the non-limitingexamples below.

EXAMPLES Example 1

Method and Material

1.1 Construction of Vectors Expressing SIRPα-Fc and SIRPαD1-FcRespectively

Plg-Tail (R&D Systems) was employed as the expression vector. The codingsequences of extracellular domains of SIRPα and SIRPαD1 were amplifiedfrom THP-1 (ATCC(TIB-202™) cells, respectively, using primer 1 (SEQ IDNO.: 9) with primer 2 (SEQ ID NO.:10), and primer 1 (SEQ ID NO.: 9) withprimer 3 (SEQ ID NO.: 11). The PCR products were cloned intoHindIII/EcoRI site in an engineered Plg-Tail, thus generating pSIRPα-Fcand pSIRPαD1-Fc expression vectors.

1.2 Construction of Vectors Expressing HY03M and HY03MM Respectively

The coding sequence of SIRPαD1 having N89A mutation was synthesized byNanjing Jinsirui biotechnology Co., Ltd (Program NO.: 7009323-1) andthen cloned into HindIII/EcoRI site of Plg-Tail vector to generate anHY03M expression vector. Using primer 4 (SEQ ID NO.: 12) and primer 5(SEQ ID NO.: 13), the nucleotide sequence (GAC) encoding aspartic acid,the 192th amino acid residue of HY03M at the Fc terminus, was mutated toencode alanine (GCC) through site-directed mutagenesis, thus generatingan HY03MM expression vector.

Table 1 PCR Primers

TABLE 1 PCR primers Primer No. Primer sequences (5′-3′) GeneEndonuclease Primer 1: CCCAAGCTTGGGGCCACC

SIRPα HindIII (SEQ ID No.: 9) Primer 2: CGGAATTC

SIRPα EcoRI (SEQ ID No.: 10) Primer 3: CGGAATT

SIRPα EcoRI (SEQ ID No.: 11) Primer 4:GAGGTCACATGCGTGGTGGTGGCCGTGAGCCACGAAGACCCTG hIgG1-Fc (SEQ ID No.: 12)Primer 5: CACCACCACG CATGTGACCTCAGGGGTCCG GGAGATCATG hIgG1-Fc(SEQ ID No.: 13) Notes: Gene specific sequences were shown in italic,and endonuclease recognition sites were underlined.

2. Protein Expression and Purification

The complete cell culture media DMEM (with 10% FBS) containing CHO cellswas added into a 24-well plate, 0.5 ml per well, and the plate was keptin an incubator for 24 hours. For transfection, 0.5 μg of plasmid DNAand 2 μl of LIPOFECTAMINE™ 2000 (Cat #11668-027, invitrogen) wereseparately dissolved in 50 μl of serum-free culture media, which werethen combined and left still at room temperature for 20 minutes.Thereafter, the medium was slowly poured into wells of the plate, andthe plate was placed in the incubator for 24 hours. On the next day, 100μl of supernatant was taken and tested for protein expression byenzyme-linked immunosorbent assay (ELISA).

3. Protein Expression Assay

Protein expression was tested by ELISA. In particular, an goatanti-human IgG antibody, F(ab′)2 fragment (Biosource international inc)was dissolved in PBS buffer, which was then added into a 96-well ELISAplate, 20 ng per well. The ELISA plate was placed in a refrigerator at4′C overnight. Upon testing, the plate was blocked with blockingsolution (PBS, 0.05% TWEEN® 20 (polysorbate 20), 3% skim milk) for 1hour, and then added with diluted serum and incubated for 1 hour at roomtemperature. After washed with a washing solution (PBS, 0.05% TWEEN® 20(polysorbate 20)) for 5 times, the plate was added with horse radishperoxidase (HRP) labeled rabbit anti-human IgG antibody (JacksonImmunoResearch Lab) and was incubated at room temperature for 1 hour.After washed for 5 times, the substrate for HRP was added. Two minuteslater, the solution (1N H₂SO₄) for stopping color development was usedto terminate the chromogenic reaction. The optical density was measuredat 450 nm.

4. Screening for Stably-Expressing Cell Line

Transfected cells were subjected to concentration-increasing antibioticscreening (GENETICIN® (G-418), Cat #10131035, Invitrogen). The unstablecells were killed gradually, and survived cells were diluted and putinto five 96-well plates, 0.5 to 1 cell per well. The plates were placedin an incubator for 10-15 days. The wells each containing a single clonewere tested by ELISA, and then the cells with positive proteinexpression were propagated, and habitually cultured with serum-treeEX-CELL® CD CHO culture media (Cat #14361C-1000ML, SIGMA). After furtherscreening, the cells with the highest expression levels were selectedand frozen for use.

5. Protein Production and Purification

The stably-expressing cell line (3×10⁵/ml) was inoculated into a 2 Lshake flask containing 300 ml of serum-free culture medium, and theshake flask was placed in a shaking bed for culture. After a celldensity of 5×10⁶/ml was reached, the supernatant was collected. Thesupernatant was purified using Protein A column. The purified proteinwas transferred to PBS (pH 7.0) with dialysis. Protein electrophoresiswas employed to provide proteins having a purity of at least 98%.

6. Target Binding Activity

The binding activities of SIRPα-Fc, SIRPαD1-Fc, and HY03M to CD47 weretested by using flow cytometry.

Two cell lines, PC-3 (human prostate cancer) and Jurkat (T lymphocyteleukemia), were used to test the binding activity of each protein toCD47. After washed with PBS, the cells were suspended in PBS with aconcentration of 1×10⁶/ml. The cell suspension was added with hlgG (1μg/ml) and then incubated in a refrigerator at 4C for 1 hour. Afterwashes with PBS, the cells were transferred to a 96-well U-shaped cellculture plate (Cat #163320, NUNC), 100 μl per well. Then, the cells wereadded with purified proteins with different concentrations and thenincubated in a refrigerator at 4C for 1 hour. The cells were washed withand then suspended in PBS. Thereafter, the cells were incubated togetherwith FITC-labeled anti-human IgG-Fc antibody (Cat #F9512, Sigma). After1 hour, the cells were tested in a flow cytometry (Guava easyCyte6HT-2L, Millipore).

7. Target Blockade Assay

In order to test whether the purified proteins can block the binding ofCD47 to SIRPα, FITC-labeled SIRPα-Fc (Cat #4546-SA-050, R&D Systems, 100nM) was mixed with unlabeled SIRPα-Fc, SIRPαD1-Fc, HY03M, HY03MM orhIgG-Fc of different concentrations. Then, each mixture was put in a96-well U-shaped plate with Jurkat cells, and incubated in arefrigerator at 4C for 1 hour. After washes with PBS, the cells werere-suspended in 200 ml of PBS and analyzed for the ratio of fluorescentcells in a flow cytometry.

8. Binding to FcγRs

The binding activities of proteins of the present invention to FcγRswere tested by ELISA as follows.

CD64 (FcγRI) (Cat: 1257-FC-050, R&D Systems), CD32a (FcγRIIa) (Cat:1330-CD-050/CF, R&D Systems), CD32b (FcγRIIb) (Cat: 1875-CD-050, R&DSystems), and CD16a (FcγRIIIa) (Cat: 4325-FC-050, R&D Systems) werediluted with a coating buffering solution (CBS) (Sigma-Aldrich Co.,Product code: 1001329288 C3041-100CAP) to a concentration of 1000 ng/ml,and 100 μl of each solution was added into an ELISA plate (Cat #442404,Nunc™), 100 ng per well. The plate was left in a refrigerator at 4Covernight. Immediately before the test, the plate was washed by 0.05%PBS-T and then blocked by 3% skim milk for 1 hour at room temperature.The diluted solutions of HY03M and HY03MM (800, 400, and 200 nM) wereadded into the plate, 100 μl per well. After incubated at roomtemperature for 1 hour, the liquids in the plate were discarded. Theplate was washed by 0.05% PBS-T for 5 times, and then added with 100 μlof HRP-Rabbit anti-human IgG Fc (Cat #: 309-036-008, JacksonImmunoResearch Lab) diluted at 1:20000. The plate was incubated at roomtemperature for 1 hour and then washed by the washing solutions for 5times. Thereafter, HRP substrates were added to the plate, and the platewas left for colorimetric reaction for 10 to 20 minutes in the dark. Thecolorimetric reaction was terminated later using 1 N H₂SO₄, and theOD450 value was obtained in a plate reader.

9. Phagocytosis Assay

Mouse macrophages (Raw264.7) were added into a 96-well plate, 5×10⁵cells per well, and incubated for 2 hours in an incubator at 37° C.Jurkat cells labelled with CFSE (2.25 μM) were incubated with 2.5 μg/mlHY03M, HY03MM or IgG-Fc for 30 minutes at 37C and then transferred tothe plate containing Raw264.7 macrophages mentioned above. The plate wasincubated at 37C for another 3 hours. With washes by PBS for 3 times,Jurkat cells free in the solution were removed. The Raw264.7 cells werethen observed in a flow cytometry through the CFSE contained in thesecells.

10. Antitumor Assay

The in vivo antitumor activity of HY03M was studied in a HL-60subcutaneous tumor model. Twenty Balb/c nude mice were injectedsubcutaneously with leukemia (HL60) cells, 4×10⁶ cells per mouse. Whenthe tumors grew up to 100 to 150 mm³ in volume, the mice were randomlydivided into 3 groups. The first group was intraperitoneally injectedwith PBS, while the second group was intraperitoneally injected with aVEGF inhibitor. The third group was intraperitoneally injected withHY03M. Each group was administered with said agent for 6 times at a doseof 10 mg/kg, twice a week. The volume and weight of the tumors weremeasured twice a week.

In order to know whether the Fc region contributed to anti-tumor effect,the anti-tumor activities of HY03M and HY03MM were tested using alymphoma model, respectively, in comparison with Rituximab. Thirty-eightBalb/c rude mice were subcutaneously injected with Daudi cells, 1×10⁷cells per mouse. When the tumors grew up to 100 to 150 mm³ in volume,the mice were randomly divided into 5 groups. The first group wasintraperitoneally injected with PBS, while the second to fifth groupwere intraperitoneally injected with HY03M, HY03MM, Rituximab, andHY03MM plus Rituximab, respectively. Each group was administered withsaid agent for 8 times at a dose of 5 mg/kg, twice a week. The volumeand weight of the tumors were measured twice a week.

EXPERIMENTAL RESULTS

1. Construction of Expression Vectors

The structure of SIRPαD1-Fc was shown in FIG. 1B and FIG. 1D, whereinSIRPαD1 was linked to the N-terminus of IgG1-Fc. The nucleic acidsequence and amino acid sequences of each protein were shown in FIG. 2to FIG. 5. The coding sequence of SIRPα-Fc consisted of 1752 nucleotides(FIG. 2A, SEQ ID No.:1), encoding 583 amino acids (FIG. 2B, SEQ IDNo.:2). Among the 1752 nucleotides, 1047 encoded SIRPα, 696 encoded Fc,and the remaining 6 formed EcoRI site. The coding sequence of SIRPαD1-Fcconsisted of 1131 nucleotides (FIG. 3A, SEQ ID No.:3), encoding 376amino acids (FIG. 3B, SEQ ID No.:4), wherein, 426 nucleotides encodedSIRPαD1, 696 encoded Fc, and the remaining 6 formed EcoRI site. HY03Mand HY03MM contained 1125 nucleotides, wherein HY03M had a N89A mutation(FIGS. 4A and 4B, SEQ ID No.:5 and 6), while HY03MM contained a N89Amutation and a D192A mutation (FIGS. 5A and 5B, SEQ ID No.:7 and 8).

2. Protein Expression Analysis

Theoretically, the four proteins, SIRPα-Fc, SIRPαD1-Fc, HY03M andHY03MM, had molecular weights of ˜128 kDa, ˜82.7 kD, ˜82.3 kDa and ˜82.3kDa, respectively. With protein electrophoresis (SDS-PAGE), it was foundthat all the molecular weights were larger than the theoreticallypredicted ones under non-reducing conditions (FIG. 6), which might bedue to protein glycosylation at a glycosylation site in relation to anAsn residue in SIRPαD1. The irregular glycosylation at this site maylead to the presence of two bands on SDS-PAGE gel under non-reducingconditions (FIG. 6B). If the site for glycosylation was removed, as inHY03M and HY03MM, only one band was found instead of two (FIGS. 6C and6D).

3. Target Binding Activity Assay

By using flow cytometry, the binding activities of SIRPα-Fc andSIRPαD1-Fc to PC-3 cells were analyzed (FIG. 7A). It was found that thebinding activity of SIRPαD1-Fc (EC50=6.57 nM) was significantly higherthan that of SIRPα-Fc (EC50=12.63 nM). As previous studies showed thatthe glycosylation had no effect on binding of D1 to CD47 (Lee W Y etal., 2007), N89 in D1 region was mutated to A (the protein variant wasdesignated as HY03M). The binding activity of HY03M to CD47 was comparedwith that of SIRPαD1-Fc. The result indicated that HY03M with theglycosylation site removed had an evidently higher binding activity totargets (EC50=0.5 nM) than SIRPαD1-Fc (EC50=1.0 nM), as shown in FIG.7B. The study here suggested that the protein containing only D1 regionhad a better target binding activity than the protein containing wholeextracellular domains, and to remove the glycosylation site in D1further promoted the binding activity (the target binding activity:HY03M>SIRPαD1-Fc>SIRPα-Fc).

4. Target Blockade Assay

With flow cytometry, the effect of the unlabeled proteins, SIRPα-Fc,SIRPαD1-Fc, HY03M and HY03MM, on binding of fluorescently labelledSIRPα-Fc with targets was studied. The results showed that, as in FIG.8, these four proteins can all block binding of the fluorescentlylabelled protein to the target Jurkat cells in a dose dependent manner,with SIRPαD1-Fc and HY03M having the best blockade effects (see thetable on lower part of FIG. 8).

5. Effect of Binding Activity of HY03M or HY03MM with FcγRs onPhagocytosis by Macrophages

The 265^(th) amino acid residue of human IgG1-Fc, aspartic acid (D), waskey to antibody's function. If aspartic acid was converted to alanine(D265A), IgG would lose its binding activity to FcγRs (FcγRI, FcγRIIA,FcγRIIB, FcγRIIIA) (Shields R L, et al., 2001) and the correspondingADCC, CDC and the like. To confirm whether Fc part in HY03M contributedto the anti-tumor activity, the aspartic acid in Fc region of HY03M wasconverted to alanine (HY03MM, D192A, FIG. 5B), and the binding activityof HY03M to FcRs (FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA) was analyzed incomparison with HY03MM. The results showed that, as in FIG. 9A, thebinding activity of the protein variant (HY03MM) to FcRs was decreasedsignificantly, especially the binding activity to FcγRIIA, FcγRIIB andFcγRIIIA.

To test the effect of the amino acid residue mutation on macrophages'activities, fluorescently (CFSE) labelled target Jurkat cells wereincubated together with macrophages (Raw264.7) and HY03M or HY03MM. Itturned out that HY03M evidently promoted the phagocytosis by macrophagesas compared to the negative control group (IgG), and HY03MM lost such afunction and showed no difference when compared to IgG, as shown in FIG.9B.

The study suggested that the blockade of CD47-SIRPα binding by, forinstance, HY03MM, was not sufficient to induce phagocytosis of targetcells by macrophages. The interaction between Fc and FcγRs on surfacesof macrophages, when combined with the blockade of CD47-SIRPα binding,will stimulate phagocytosis.

6. In Vivo Anti-Tumor Activities of HY03M and HY03MM

The in vivo anti-tumor activity of HY03M was studied in a HL-60subcutaneous tumor model. As shown in FIG. 10A, after treated by theVEGF inhibitor, tumor growth was not evidently inhibited intumor-bearing mice. In the group with HY03M treatment, tumor growth wassignificantly inhibited, wherein the tumor size decreased gradually from100 mm³ at the beginning of treatment, and almost vanished at the end ofthe experiment. In the negative control group, tumor size increased overtime and became 1000 mm³ at the end of the experiment. The resultssuggested that the activity inhibition of VEGF alone had no obvioustreatment effect on HL60 tumor, indicating that the growth of HL60tumors did not depend much on VEGFs. However, if the inhibitory effecton phagocytosis by macrophages was removed, phagocytosis of tumor cellsby macrophages would be promoted, eliminating tumor cells.

To test whether the Fc region was involved in HY03M's anti-tumoractivity, the therapeutic effects of HY03M, HY03MM and Rituximab onlymphoma were studied using a lymphoma (Daudi) model. It can be seen(FIG. 10B) that tumor growth was significantly inhibited in the groupwith HY03M treatment (TGI=72.5%), which was much better than that inRituximab group (TGI=45.6%). However, HY03MM with Fc region having amutation had a much attenuated inhibitory effect on tumor growth(TGI=26.4%), suggesting that Fc region was involved in HY03M'santi-tumor activity.

The above data indicated that HY03M treated tumors by i) inhibitingbinding of CD47 with SIRPα so that the inhibitory signals transmitted bySIRPα were blocked and macrophages were activated; and ii) binding Fc toFcRs to active macrophages.

CONCLUSIONS

Our studies indicated that the recombinant protein SIRPαD1-Fc had a goodtarget binding activity which was better than that of SIRPα-Fc. If thesite for glycosylation was removed, as in HY03M, the target bindingactivity was further improved. In vivo studies showed that HY03M had agood anti-tumor activity, and would completely eliminate tumors in HL60model. The protein fought tumors by i) inhibiting binding of CD47 withSIRPα so that the inhibitory signals transmitted by SIRPα were blockedand macrophages were activated; and ii) binding Fc to FcRs to activemacrophages. The two mechanisms produced a synergistic effect,sufficiently stimulating phagocytosis of tumor cells by macrophages. Theactivated macrophages may further present tumor antigens to Tlymphocytes (Tseng D, et al., 2013) and kill the tumor cells finally.

As described above, for the binding activity of SIRPα-Fc or SIRPαD1-Fcto CD47, it was once reported (Lee W Y et al., 2007) that the affinitiesof these two proteins to CD47 were not different. Our studies hereshowed that the affinity of SIRPαD1-Fc to CD47 (in PC-3 cells)(EC50=6.57 nM) was much higher than that of SIRPα-Fc (EC50=12.63 nM).With amino acid analysis, it was found that SIRPαD1-Fc had 9 more aminoacid residues (SCAWSGVAG (SEQ ID NO.: 14)) at the N-terminus compared tothat constructed by Lee W Y et al., which might contribute to theincreased target binding activity. Further studies found that the targetbinding activity was further improved when the glycosylation site (N89A)in D1 region was removed. In addition, with site-directed mutagenesis(D192A) in Fc region, Fc was found to help protein purification (byProtein A chromatography) and to improve protein stability, and also beinvolved in HY03M's anti-tumor activity, as the anti-tumor activity ofthe variant protein with D192A was greatly decreased.

While the invention has been described above in connection with one ormore embodiments, it should be understood that the invention is notlimited to those embodiments, and the description is intended to coverall alternatives, modifications, and equivalents, as may be includedwithin the spirit and scope of the appended claims. All referenced citedherein are further incorporated by reference in their entirety.

REFERENCES

-   1. Gardai S J, McPhillips K A, Frasch S C, Janssen W J, Starefeldt    A, Murphy-Ulrich J E, Bratton D L, Oldenborg P A, Michalak M, Henson    P M. Cell-surface calreticulin initiates clearance of viable or    apoptotic cells through trans-activation of LRP on the phagocyte.    Cell. 2005; 123:321-334.-   2. Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P,    Zitvogel L, Kroemer G. Calreticulin exposure is required for the    immunogenicity of gamma-irradiation and UVC lightinduced apoptosis.    Cell Death Differ. 2007; 14:1848-1850.-   3. Orr A W, Pedraza C E, Pallero M A, Elzie C A, Goicoechea S,    Strickland D K, Murphy-Ullrich J E. Low density lipoprotein    receptor-related protein is a calreticulin coreceptor that signals    focal adhesion disassembly. J Cell Biol. 2003; 161:1179-1189.-   4. Theocharides. A. P. A.; Jin, L. O.; Cheng, P. Y.; Prasolava, T.    K.; Malko, A. V.; Ho. J. M.; Poeppl, A. Q.; Rooijen, N. van;    Minden, M. D.; Danska, J. S.; Dick. J.; Wang. J. C. Y. J. Exp. Med.    2012 Vol. 209 No. 10 1883-1899-   5. Lee W Y et al. Novel Structural Determinants on SIRPα that    Mediate Binding to CD47. J Immunol 2007; 179:7741-7750.-   6. Shields R L et al. High Resolution Mapping of the Binding Site on    Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1    Variants with Improved Binding to the FcgR. JBC. 2001,    276:6591-6604.-   7. Tseng D, et al. Anti-CD47 antibody-mediated phagocytosis of    cancer by macrophages primes an effective antitumor T-cell response.    PNAS. 2013, 110:11103-11108

What is claimed is:
 1. A recombinant bi-functional fusion protein,comprising an extracellular Ig-like domain of a signal-regulator protein(SIRP) of SIRPα (SIRPαD1), linked to an Fc region of an immunoglobulincomprising both CH2 and CH3 regions of the immunoglobulin, wherein: theSIRPαD1 comprises a mutation, wherein: the mutation corresponds to N toA at position 89 in full length SIRPα set forth in SEQ ID NO.: 2; andthe recombinant bi-functional fusion protein can bind to CD47 and FcγReceptor, thereby blocking the binding of CD47 to the SIRP on surfacesof macrophages and stimulating phagocytosis of tumor cells by themacrophages.
 2. The recombinant bi-functional fusion protein of claim 1,wherein the Fc fragment is an Fc fragment of IgG1.
 3. The recombinantbi-functional fusion protein of claim 2, wherein the IgG1 is a humanIgG1.
 4. The recombinant bi-functional fusion protein of claim 3,wherein the Fc region comprises an amino acid sequence having at least90% identity to the amino acid sequence set forth in SEQ ID NO: 6 fromresidue 143 to residue
 374. 5. The recombinant bi-functional fusionprotein of claim 3, wherein the Fc region comprises an amino acidsequence having at least 95% identity to the amino acid sequence setforth in SEQ ID NO: 6 from residue 143 to residue
 374. 6. A homodimer ofthe recombinant bi-functional fusion protein of claim 1, wherein thehomodimer comprises two fusion proteins linked by one or more disulfidebonds.
 7. The recombinant bi-functional fusion protein of claim 1,comprising an amino acid sequence having at least 90%, identity to theamino acid sequence set forth in SEQ ID NO.:
 6. 8. The recombinantbi-functional fusion protein of claim 1, comprising an amino acidsequence having at least 95% identity to the amino acid sequence setforth in SEQ ID NO.:
 6. 9. A polynucleotide encoding the recombinantbi-functional fusion protein of claim
 1. 10. A polynucleotide encodingthe recombinant bi-functional fusion protein of claim
 8. 11. Anexpression vector comprising the polynucleotide of claim
 9. 12. A hostcell comprising and expressing the polynucleotide of claim
 9. 13. A hostcell comprising the expression vector of claim
 11. 14. A pharmaceuticalcomposition, comprising an effective amount of the recombinantbi-functional fusion protein of claim 1 for treating a disease caused byover expression of Cluster of Differentiation 47 (CD47), and at leastone pharmaceutically-acceptable excipient, wherein the recombinantbi-functional fusion protein can bind to CD47 and Fcγ Receptor, therebyblocking the binding of CD47 to the SIRP on the cell surface ofmacrophages and stimulating phagocytosis of tumor cells by macrophages.15. A method for treating a disease caused by over-expression of CD47,comprising administering to a patient or a subject a therapeuticallyeffective amount of the pharmaceutical composition of claim
 14. 16. Themethod of claim 15, wherein the disease is selected from the groupconsisting of acute myelocytic leukemia (AML), chronic myelocyticleukemia (C1v1L), acute lymphoblastic leukemia (ALL), non-Hodgkin'slymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer,prostate cancer, lung cancer, colon cancer, breast cancer, pancreaticcancer, and renal cell carcinoma.
 17. The method of claim 15, whereinthe disease is selected from the group consisting of Crohn's disease,allergic asthma and rheumatoid arthritis.
 18. A method of preparing arecombinant bi-functional fusion protein, comprising an extracellularIg-like domain of a signal-regulator protein (SIRP) of SIRPα (SIRPαD1),linked to an Fc region of an immunoglobulin comprising both CH2 and CH3regions of the immunoglobulin, wherein: the SIRPαD1 comprises amutation, wherein: the mutation corresponds to N to A at position 89 infull length SIRPα set forth in SEQ ID NO.: 2; and the recombinantbi-functional fusion protein can bind to CD47 and Fcγ Receptor, therebyblocking the binding of CD47 to the SIRP on surfaces of macrophages andstimulating phagocytosis of tumor cells by the macrophages; said methodcomprising cultivating host cells of claim 13 to express the protein andpurifying the protein.
 19. A host cell comprising an expression vectorcomprising the polynucleotide of claim 8; wherein said host cellexpresses the polynucleotide.
 20. A method of preparing a recombinantbi-functional fusion protein, comprising an extracellular Ig-like domainof a signal-regulator protein (SIRP) of SIRPα (SIRPαD1), linked to an Fcregion of an immunoglobulin comprising both CH2 and CH3 regions of theimmunoglobulin, wherein: the SIRPαD1 comprises a mutation, wherein: themutation corresponds to N to A at position 89 in full length SIRPα setforth in SEQ ID NO.: 2; and the recombinant bi-functional fusion proteincan bind to CD47 and Fcγ Receptor, thereby blocking the binding of CD47to the SIRP on surfaces of macrophages and stimulating phagocytosis oftumor cells by the macrophages; said method comprising cultivating hostcells of claim 19 to express the protein and purifying the protein.